Anal. Chem. 1909, 65,617-622
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Dehydrogenase-Modified Carbon-Fiber Microelectrodes for the Measurement of Neurotransmitter Dynamics. 1. NADH Voltammetry Werner G. Kuhr,' Valerie L. Barrett? Michelle R. Gagnon, Phillip Hopper, and Paul Pantano Department of Chemistry, University of California, Riverside, California 92521
The vottammetry of NADH has been characterized at carbonfiber microelectrodes at scan rates up to 100 V/s. Eiectrochemicalpretreatmentof the electrode dramatically changed the propertlor of the modified electrode. Anodic pretreatment of the surface resulted in an adsorptive wave for NADH oxidation, while le88 adsorptlon was evident under more modorate condttions. The pH of the buffer used for the anodlzatlon played a critical role in determinlng the voltammet& peak shape. Oxidation of NADH at slow scan rates (lo0 V/s) at carbon-fiber microelectrodes has been used extensively to monitor the release and uptake of the easily-oxidized neurotransmitters (i.e. dopamine) in vivo.lV2 Unlike the catecholamines,most other neurotransmitters and metabolic intermediates are not electroactive at analytically useful potentials. In fact, most of these molecules have very few physical properties which can be utilized for chemical analysis (i.e. visible or ultraviolet absorption, fluorescence, or electrochemicalactivity). Chemical derivatization of these molecules requires extensive sample handling, and this requires the expenditure of time to carry out the physicalhandling and derivatization reactions. Thus, the small quantities and low concentrations of analyte limit the utility of derivatization methods for in vivo measurements, especially when the analysis must be performed on a second or millisecond time frame. One possible solution to this problem is the use of an enzyme-modified microelectrode. Even though there are many "biosensors" reported in the literature? relatively few have the requisite size, sensitivity, selectivity, and temporal resolution to make such measurements. Additionally, most of these biosensors use oxidases, which liberate or consume oxygen. Since oxygen levels vary dramatically during neurotransmission,4it can be difficult to dissociate physiological fluctuations from the oxidase-generated changes in oxygen concentration. This problem might be alleviated through + Present address:
Sunkist Growers, Inc., 760 E. Sunkist St., Ontario,
CA 91761.
(1) Kuhr, W. G.; Wightman, R. M. Brain Res. 1986, 381, 168-171.
(2) May, L. J.; Kuhr, W. G.; Wightman,R. M. J . Neurochem. 1988,51,
1 - -0- -M -9-..
(3) Turner, A. P. F.; Karube, 1.; Wilson, G. S. Biosensors; Oxford University Press: Oxford, 1987, 770 pp. (4) Zimmerman, J. B.; Wightman, R. M. Anal. Chem. 1991,63,24-28. 0003-2700/93/0365-0617$04.00/0
the use of other enzymes coupled to different redox equilibria Very few biosensor reporta involve the use of dehydrogenasecatalyzed reactions, which are linked to the NAD+/NADH redox couple. This is because of the difficulty in the electrochemical measurement of NADH."' The electrochemical oxidation of NADH shows a large overpotential at most electrode materials (the El/*for the electrochemicaloxidation5.7 is typically 0.50 to 0.80V v8 SCE comparedto the Eo calculatedfor the homogeneousreaction,whichis-O.56V v8 SCE). Additionally,the products of NADH oxidation have been shown to adsorb strongly on these electrodesand passivatethe electrode surface.8 This problem is exacerbated when NAD+ is present; the electrode surface is fouled very quickly, albeit consistently.6 Many attempta have been made to reduce this large overpotential for NADH oxidation through use of immobilized redox mediators.QJ0 The problem with this approach is one of selectivitrcan its oxidation potential be decreased selectively, so that other easily oxidized species are not measured? This is difficult, since most of the protocols which reduce the overpotential for NADH oxidation will do the same for other interferenta (e.g., ascorbic acid, uric acid, DOPAC, etc.). Voltammetry at ultramicroelectrodes has several advantages over that performed at larger electrodes.11 Electrodes of micrometer dimensions have extremely low charging currents and fast response times. This facilitates the use of faster voltammetric scan rates (>lo0V/s) which allows one to take analytical data with better temporal resolution. Additionally, fast scan techniques can differentiate species on the basis of their electron-transfer kinetics as well aa their redox potentials. Species which have fast electron-transfer kinetics show reversiblevoltammetryat fast sweep rates, while less reversiblespecies exhibit larger and larger overpotentiale and, consequently, are easily distinguished from the more reversible species. This strategy has been very effective in minimizing interferences for the measurement of dopamine in vivo, where nanomolar levels can be measured selectively in the presence of a 10 000-fold excess of ascorbic acid.2J2 Since the rate of heterogeneous electron transfer for the oxidation of NADH is fast, the same strategy should be useful for the measurement of NADH. Another advantage of these (5) Blankespoor, R. I.; Miller, L. L. J. Electroanal. Chem. Interfacial Electrochem. 1984,172,231-241. ( 6 ) Moiroux, J.;Elving, P. J. Anal. Chem. 1979,49,346-350. Moiroux, J.; Elving, P. J. J. Am. Chem. SOC.1980, 102, 6633-38. (7) Samec, Z.; Bresnahan, W. T.; Elving, P J. J . Electroanal. Chem. Interfacial Electrochem. 1982, 133, 1-23. (8) Moiroux, J.; Elving, P. J. A w l . Chem. 1978,50,1066-62. Cenas, N. K.; Kanapieniene, J. J.; Kuls, J. J. J. Electroanal. Chem. Interfacial Electrochem. 1985, 189, 163-169. (9) Gorton, L.; Torstensson, A.; Jaegfeldt, H.; Johansson, G. J. Electroanal. Chem. Interfacial Electrochem. 1984,161, 103-120. (10) Gorton, L. J. Chem. SOC.,Faraday Tram. 1 1986,82,1245-1258. (11)Wightman, R. M.; Wipf, D. 0. J.Electroanal. Chem. Interfacial Electrochem. 1989, 15, 267-353. (12) Stamford, J. A.; Hurst, P. R.; Kuhr, W. G.; Wightman, R. M. J. Electrochem. Chem. Interfacial Electrochem. 1989, 265, 291-296. 0 1993 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
Scheme
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fast electrochemical measurements is that electrode passivation is minimized. Several electrochemical pretreatments were examined to optimize the quality and reproducibility of the voltammetric measurement. Anodic pretreatments at neutral or basic pH were found to increase adsorption of NADH/NAD+. This adsorption was short-lived, in that continuous application of the 100 V/s waveform resulted in diminution and eventual disappearance of the adsorbed wave. The same anodic pretreatment in acidic solution yielded a more reproducible, diffusion-controlled profile for the oxidation of NADH at 100 V/s. The voltammetric response was linear from 1mM NADH to the detection limit of 7 pM. The response time for this measurement was determinedwith flow injection analysis to be 184 f 65 ms, which is as fast as could be measured with this system. A microdialysis electrode was constructed where the enzyme, glutamate dehydrogenase, was entrapped within a 150-pm4.d. hollow microdialysis fiber. This served as a prototype biosensor for the measurement of enzyme-generated
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NADH. EXPERIMENTAL SECTION Chemicals. Glutamate dehydrogenase (GDH), 40 units/mg, (EC 1.4.1.3), @-nicotinamide adenine dinucleotide (NAD+), @-nicotinamideadenine dinucleotide, reduced form (NADH), L-glutamic acid (Sigma); Epon 828 Resin (Shell Oil) and 1,4phenylenediamine (Aldrich) were used without further purification. The phosphate buffer consisted of 150 mM sodium chloride and 100 mM sodium phosphate heptahydrate, adjusted to pH 7.4 unless otherwiseindicated. All solutions were prepared in water degassed with nitrogen and purified by a Milli-Q water purification system (Millipore). Carbon-Fiber Microelectrodes. Fabrication of carbon-fiber microelectrodes(Thornel P-55S, Amoco Performance Products) has been described pre~ious1y.I~ Briefly, a single 10-pm-diameter carbon fiber is aspirated into a glass capillary tube (1.2-mm o.d., 0.68-mm i.d., A-M Systems No. 60201, and a tapered end is produced after the capillary tube is pulled by a Narishige Model PE-2 microelectrode puller. A seal between the fiber and the capillary tube is achieved by utilizing Epon 828 epoxy with 12% by weight 1,4-phenylenediamineas hardener. Initial activation of the carbon surface was achieved by polishing for 10 min with diamond paste (1 pm, Buehler). The polished electrode was sonicated in hot toluene for 10 s and deionized water for 10 s to remove residual polishing material. Electrochemical pretreatmenta consisted of a 3-8 application of a 50-H~cyclic potential waveform (initial potential, -0.2 V), with a switching potential of either 1.8 or 2.0 V. All potentials were referenced versus a
(13)Kelly, R.S.;Wightman, R.M.Anal. China. Acta 1986,187,79-87.
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Potential (mV vs. Ag/AgCl) Flgure 1. NADH voltammetry at carbon-fiber microelectrodes. Top:
Cyclic staircase voltammetry at 1 VIS was repeated at a polished
carbon-fiber microelectrode with a 4 8 interval between scans for 1 mM NADH. The first (-), tenth (.), and hundredth (+++) scans obtained are shown. Bottom: Cyclic staircase voltammetry at 100 V I S was repeated at a poltshed carbon-fiber microelectrode with a 200-ms Interval between scans in 1 mM NADH. The fkst (-), tenth (+++),and thousandth (-) backqwnbsubtractedsans are shown.
Ag/AgCl reference electrode. The pretreatment was performed in either pH 12 phosphate, pH 7.4 phosphate buffer, or 1.0 M HC1. Instrumentation. Cyclic staircase voltammetry was performed with an EL400 potentiostat (Ensman Instruments, Bloomington, IN) where all waveforms were generated and currents acquired via an 80386personal computer using an A/DD/A interface (Labmaster DMA, ScientificSolutions,Solon,OH). A 100-MHzdigital oscilloscope (Hewlett-Packard 54501-A)was used to observe all transients, and a Hewlett-Packard ColorPro was used to plot data. The flow injection analysis system consisted of a pneumatic actuator (Rheodyne, Model 5701) controlled via a solenoid valve (Rheodyne kit, Model 7163). A locally-built solenoid driver circuit allowed computer control of the compressed air-driven valves which switched flow from the buffer loop to the sample loop of the FIA. The electrochemical
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1903 019 Not Pretreated. 0
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Flgurr 2. Effect of electrode pretreatment of NADH voltammetry. Various electrochemical pretreatments were applled to polished, carbon-fiber microelectrodesto Increase the reverslbliky of NADH voltammetry. Voltammetryof NADH (1 mM) at 100 VIS Is showfor a serles of three electrodas each under the following conditions: (A) polished electrode; (B) a cyclic potentiel waveform between -0.2 and 2.0 V vs Ag/AgCl at 50 Hz was applled for 3 s In pH 7.4 phosphate buffer; (C) a cyclic potential waveform between -0.2 and 2.0 V vs Ag/AgCI at 50 Hz was applled for 3 s In pH 12 phosphete; (D) a cyclic potential waveform between -0.2 and 2.0 V vs AglAgCl at 50 Hz was applled for 3 s In 1 M HCI.
cell was constructed from Plexiglas in such a way as to allow the microelectrodeto be positioned ca. 60mm from the output of the FIA sample loop. This cell was designed to match the internal diameter of the FIA tubing (0.75 mm) in order to minimize diffusional broadening of the analyte as it was transported to the microelectrode (specifications critical for the measurement of a subsecond response time). Finally, the flow of buffer (0.81 mL/ min) was controlled by a syringe pump (SageInstruments Model 341-B) or by gravity flow. Construction of a Microdialysis Electrode. T w o procedures were used to secure a dialysis fiber to the end of a carbonfiber microelectrode (Scheme I). A 2-cm length of 150-pm-i.d. hollow dialysis membrane (9OOO MWCO, Spectrum Medical Industries, Los Angeles, CA) was attached to an empty glass capillary pipet with 5-min epoxy. The dialysis fibers were then flushed with ethanol for 10 min and with deionized water for 30 min. Next, a suspension of glutamate dehydrogenase was transferred into the membrane. The free end of the enzymefilled membrane was then carefully guided onto the polished carbon-fiber microelectrodetip and attached with epoxy. When dry, the membrane was cut with a scalpel approximately 500 Mm from the end of the electrode tip and sealed with epoxy. Alternatively, the dialysis fiber (length ca. 1cm) was cleaned with ethanol for 10min, followed by deionized water for 30 min. The fiber was placed onto a microscope slide under a stereo microscope (Bauschand Lomb) and allowed to dry. Asuspension of glutamate dehydrogenasewas dialyzed for 24 h (12 000-14 OOO MWCO,Spectrum) versus phosphate buffer to remove excess salts. One drop of the dialyzed solution was placed at one end of the fiber; capillary action fiied the fiber with the enzyme solution. The fiber was then allowed to dry, and the technique was repeated to increase the enzyme concentration inside the fiber. Apolished carbon-fiber microelectrodewas mounted onto a three-dimensional stereotaxic manipulator (KopfInstruments, Tujunga, CA) and carefullypositioned into one end of the dialysis fiber. The fiber was cut approximately 0.2 mm beyond the electrode tip, and epoxy was applied to both the electrode/fiber junction and the open end of the attached dialysis membrane to
prevent outward diffusion of the enzymeonce placed in solution. The distance betweenthe epoxy at the fiber end and the electrode tip was minimized to reduce longitudinal diffusion of NADH to the electrode surface. The final assembly was allowed to cure in air for 45 min and then was immersed in a 12% v/v EtOH/ phosphate buffer solution to facilitaterehydration of the dialysis fiber. Once hydrated, the electrode was transferred to a buffer solution containing 1mM NAD+ for 30 min, then immersed in the FIA flow cell and flushed with fresh buffer (containing 1mM NAD+)for 10 min. Both methods of construction were equally successful, although the latter was considerably more efficient. All sensors were stored in phosphate buffer at 0-5 OC to prevent the dialysis membrane from drying.
RESULTS AND DISCUSSION Voltammetry of NADH at Carbon-Fiber Microelectrodes. The half-wave potential (&p) for the oxidation of NADH at a polished carbon-fiber microelectrode is approximately 500 mV vs Ag/AgCl (Figure 1, top), which is very similar to that observed at macroscopic glassy carbon electrodes."' This represents at least a 1-V overpotential as compared to the EO calculated for the homogeneous reaction (-0.56 V vs SCE),699 A large overpotential can indicate slow electron-transfer kinetics, but this is certainly not the case for the oxidation of NADH at carbon fibers. As shown in Figure 1(bottom), the voltammetry is quite electrochemically reversible at scan rates up to 100 V/s. This is interesting to note, since many previous investigators have ascribed this large overpotential to slow electron-transfer kinetics. From these data, it is easy to see that electron-transfer kinetics are not responsible for the observed overpotential. There is considerable variability in the reversibility of NADH oxidation at different carbon-fiber electrodes as demonstrated by voltammetry at 100 V/s (Figures 1and 2). A clean, polished carbon-fiber surface can produce an
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
oxidativewave with a diffusive peak potential (E,) anywhere between 600 and lo00 mV vs Ag/AgCl (Figure 2A). Though there can be great variation from one electrode to another, the voltammetry of NADH at the same electrode is very consistent. A mild electrochemical pretreatment was employed to improve the reversibility of the voltammetry, since severalinvestigatorshave reported that the presence of oxygen functionalities on carbon facilitates electron transfer with NADH.14-16 Oxidation in a neutral or basic pH buffer decreases the overpotential for the diffusion-controlledwave but also introduces a very sharp, symmetrical prepeak at roughly 500 mV, which is presumably due to product adsorption (Figure 2B).* This pretreatment decreases the reproducibility of NADH voltammetry from electrode to electrode, in that the magnitude of the peak current and potential of both diffusion-controlledand adsorptive waves are extremely variable (Figure 2B,C). Adsorption of an analyte onto an electrode surface has often been used to increase the sensitivity of an electrochemical measurement.2 This has been especially true for the measurement of the catecholamine neurotransmitters, where fast adsorption kinetics allow preconcentration of dopamine onto the electrode surface, resulting in greatly enhanced detectionlimits without loss of temporal response.lP2 In the case of NADH, adsorption could serve the same function, i.e., to improve detection limits. However, the magnitude of NADH adsorption was found to diminish as a function of the number of voltammetric scans applied to an electrode. As shown in Figure 3 (top),the diffusion-controlled component for the oxidation of 100 pM NADH was virtually unchanged over 2850 scans, while the adsorptive wave at 525 mV gradually disappeared as the electrode was continuously scanned in pH 8.5 buffer. Diffusion-controlledbehavior could be obtained at any of these surfaces simply by cycling the electrode in buffer for a few thousand scans at 100V/s. Once this was done, very reproducible voltammetry could be obtained at the electrode. This is demonstrated by the concentration independence of NADH voltammetry (Figure 3, bottom). As shown, the voltammetry at 100 V/s is independent of concentration in the diffusion-controlled region (>750 mV), but some adsorption of NADH is apparent at low concentrations. The time course for the appearance of both diffusion-controlledand adsorptive waves was identical (
